Tumor animal model construction method for simulating clinical metastasis characteristics

Abstract

The invention discloses a tumor animal model construction method for simulating clinical metastasis characteristics, and relates to the technical field of biological medicine. Comprising the following steps: S1, modifying a CXCR4 chemokine receptor of a tumor cell: modifying a target tumor cell by adopting a CRISPR-Cas9 technology; high-efficiency targeting of tumor metastasis is realized through double mechanisms of CXCR4 gene precise modification and target organ microenvironment directional regulation and control: on one hand, a CRISPR-Cas9 technology is used for editing a CXCR4 gene key functional region, so that the receptor expression quantity is stably increased by 2-5 times, and the chemotactic response capability of tumor cells is enhanced; on the other hand, recombinant human SDF-1alpha is locally injected, a specific chemotactic signal gradient is constructed for a target organ, the final model can achieve the effects that the number of target organ transfer lesions is larger than or equal to 5 / organ, the non-target organ transfer rate is smaller than or equal to 10%, the clinical transfer rule is strictly followed, the clinical'primary lesion-target organ 'transfer path is accurately reengraved, and the target organ transfer rate is smaller than or equal to 10%. And the defects of random transfer and poor target organ specificity of the traditional model are thoroughly improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for constructing a tumor animal model that simulates clinical metastasis characteristics. Background Technology

[0002] Tumor metastasis is a core cause of treatment failure and poor prognosis in clinical cancer patients. Constructing animal models that accurately simulate the characteristics of clinical tumor metastasis is a crucial tool for elucidating metastasis mechanisms, screening anti-metastatic drugs, and advancing personalized treatment research. Currently, commonly used methods for constructing tumor animal models mainly include tail vein injection metastasis models, orthotopic seeding spontaneous metastasis models, and immunodeficient mouse tumor-bearing models. However, these methods have significant limitations in simulating clinical metastasis characteristics and cannot meet the needs of clinical translational research.

[0003] In practical applications, existing tumor animal model construction technologies have the following key problems: ① Poor metastasis targeting, unable to accurately simulate the clinical "primary lesion - specific target organ" targeted metastasis pattern, with target organ metastasis rate generally below 30% and non-target organ random metastasis rate exceeding 20%; ② Insufficient model stability, lack of standardized procedures for cell pretreatment, gene modification, etc., such as cell viability control and screening of metastasis-related receptor expression without clear standards, resulting in a coefficient of variation of more than 30% in the number of metastatic lesions in different batches of models, and low reliability of experimental results; ③ Weak clinical relevance, with the expression levels of core clinical metastasis markers such as VEGF and MMP-9 in metastatic lesions differing from those in primary lesions by less than 1 time, and mostly relying on immortalized cell lines, lacking the individualized characteristics of primary cells from patients, and unable to match the molecular pathological mechanisms of clinical tumors.

[0004] In view of this, we propose a method for constructing a tumor animal model that simulates clinical metastasis characteristics, in order to solve the bottleneck problems of poor orientation, low stability and insufficient clinical relevance of existing technologies, and to provide an experimental vehicle that is more in line with clinical practice for tumor metastasis research. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a tumor animal model that simulates clinical metastasis characteristics, thereby solving the technical problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for constructing a tumor animal model that simulates clinical metastasis characteristics, comprising the following steps: S1. CXCR4 chemokine receptor modification of tumor cells: Target tumor cells were modified using CRISPR-Cas9 technology, with the editing target being the 120-150bp coding region of the CXCR4 gene NM_001308142.1 sequence. After modification, CXCR4 expression increased 2-5 times compared to before modification, as detected by flow cytometry, with unmodified cells as a control. S2. In situ seeding of modified tumor cells: The cells from step S1 were adjusted to a concentration using sterile PBS containing 0.2% trypan blue at pH 7.2-7.4, and then in situ seeded into the primary lesions of 6-8 week old NOD / SCID mice or BALB / c nude mice. Specifically: When the target organ for metastasis is the brain, and the primary lesion is the lung, intratracheal injection is used, with an injection depth of 2-3 mm and a cell seeding quantity of 1×10⁶ cells. 6 -5×10 6 Inoculation volume: 50-100 μL / animal; When the target metastatic organ is the liver and the primary lesion is the colon, submucosal injection into the intestinal wall is used, with an injection depth of 1-2 mm. The number and volume of cells inoculated are consistent with the brain metastasis model. S3. Target organ microenvironment-directed regulation: Two days after inoculation, a specific ligand of CXCR4 was injected locally into the target transfer organ. The ligand was recombinant human SDF-1α, with a concentration of 50-80 ng / mL and a single injection volume of 30-40 μL / animal. The injection frequency was once every 2 days, for a total of 3 injections. S4. Model Cultivation and Validation: Animals treated in step S3 were cultured for 3 weeks in an SPF-grade environment with a temperature of 22-25℃, humidity of 40-60%, and a 12-hour light-dark cycle. Animals were weighed weekly during the cultivation period. Fluorescence signals in the target metastatic organs were detected using in vivo imaging, and the number of metastatic lesions was counted using HE staining. Immunohistochemistry was used to detect the expression levels of VEGF and MMP-9 in the metastatic lesions. A model was considered a qualified model if the following conditions were met: The number of metastatic lesions in the target organ is ≥5 per organ, and the metastasis rate in non-target organs is ≤10%; The H-score values ​​of VEGF and MMP-9 in metastatic lesions were 1.5-3 times higher than those in primary lesions. The H-score was quantitatively calculated using ImageJ software. The staining intensity was divided into three grades: weak, medium, and strong according to the OD value. The proportion of positive cells was graded according to the counting results.

[0007] Preferably, the method further includes tumor cell pretreatment, specifically as follows: Target tumor cells are seeded in high-glucose DMEM medium containing 10% mycoplasma-negative fetal bovine serum and 1% penicillin-streptomycin, wherein the penicillin concentration is 100 U / mL and the streptomycin concentration is 100 μg / mL; the culture environment is a constant temperature incubator at 37°C and 5% CO2; when the cell confluence reaches 80-90%, the cells are digested with 0.25% trypsin-EDTA digestion solution at 37°C for 2.5 minutes; then centrifuged at 1100 rpm for 6 minutes, and the cells are collected and resuspended in sterile PBS buffer at pH 7.2-7.4; the cell concentration is adjusted to 5 × 10⁶ cells / mL. 7 -8×10 7 Cell viability was measured by trypan blue staining, with a requirement of ≥90% live cells. Cells were then used for future reference.

[0008] Preferably, the CRISPR-Cas9 technology operation in step S1 specifically includes the following steps: sgRNA Design and Validation: An sgRNA targeting the CXCR4 gene was designed with a forward sequence of 5'-GGAATGGCGTGGATGAAGCG-3' and a reverse sequence of 5'-CGCTTCATCCACGCCATTCC-3'. The purity of the sgRNA was verified by 1% agarose gel electrophoresis, requiring a purity of ≥98%. Vector construction: The sgRNA and Cas9 protein-coding sequence were constructed into the lentiviral vector pLVX-EF1α-Puro. The GenBank accession number for the Cas9 protein-coding sequence is NM_001191186.1, and the Addgene number for the lentiviral vector pLVX-EF1α-Puro is 85132. During vector construction, the restriction endonuclease BsmBI was used for digestion at 37°C for 2 hours, followed by ligation with T4 DNA ligase at 22°C for 1 hour. The ligation product was transformed into DH5α competent cells, plated on LB agar plates containing ampicillin, and cultured at 37°C for 16 hours. Single clones were picked for sequencing verification. The primers used for sequencing were U6-F and U6-R. Lentiviral Packaging and Transfection: The sequencing-verified vector was transfected into 293T cells to package lentivirus. One day before transfection, 293T cells were seeded into 6-well plates at a seeding density of 2 × 10⁶ cells / well. 6 Transfection was performed using vector and Lipofectamine 3000 1 μg: 2 μL at a ratio of 1 μg to 2 μL. The medium was incubated at 37°C for 6 hours, then replaced with fresh medium. Viral supernatant was collected 48 hours after transfection and filtered through a 0.45 μm filter. Viral titer was determined using the CCK-8 assay, requiring a titer ≥ 1 × 10⁻⁶. 8 TU / mL, titer calculation was based on the viral dilution at 50% viability of 293T cells; Cell selection: Lentiviral cells were added to the target tumor cell culture system at MOI=15, along with 8 μg / mL polybrene, and incubated at 37°C for 24 hours. After 48 hours of transfection, the medium was replaced with fresh medium. After 72 hours, the cells were selected with 2 μg / mL puromycin for 7 days, with the puromycin-containing medium being replaced daily during the selection period. Cell lines expressing high CXCR4 were selected by flow cytometry, with untransfected cells as a negative control. Cell lines with fluorescence intensity 3-4 times higher than the negative control were selected and cultured to the logarithmic growth phase for later use.

[0009] Preferably, the target tumor cells in step S2 are selected from any of the following: The non-small cell lung cancer cell line is A549, and its ATCC number is CCL-185. HCT116 colorectal cancer cells, ATCC designation CCL-247; The primary cells derived from the patient underwent the following processing: fresh lung cancer or colorectal cancer tissue with a volume ≥0.5 cm³ was obtained with the patient's informed consent; the tissue was washed three times with PBS containing penicillin (100 U / mL) and streptomycin (100 μg / mL) for 5 minutes each time; the tumor tissue was minced to less than 1 mm³, and 0.1% collagenase IV was added, followed by shaking digestion at 37°C and 120 rpm for 45 minutes; the cells were then filtered through a 70 μm cell sieve to obtain a single-cell suspension. The culture medium was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were cultured in DMEM / F12 medium containing 15% fetal bovine serum and 2 ng / mLEGF at 37°C and 5% CO2. The medium was changed every 2-3 days during culture, and the number of passages did not exceed 5. The purity of the tumor marker CK18 was confirmed by flow cytometry, and the proportion of CK18 positive cells was required to be ≥95%. At the same time, the cell identity was verified by short tandem repeat sequence detection to ensure that the STR classification was consistent with the original tumor tissue.

[0010] Preferably, the chemokine ligand SDF-1α in step S3 needs to meet the following verification criteria: Purity verification: SDS-PAGE electrophoresis was performed using a 12% separating gel at 100V for 90 minutes. After Coomassie Brilliant Blue staining, the proportion of the SDF-1α band gray value to the total band gray value was analyzed using ImageJ software. This proportion was required to be ≥95%. Endotoxin detection: The Limulus amebocyte lysate (LAL) reagent method was used for detection. The procedure was followed according to the kit instructions. The standard endotoxin concentration was used as a control for calculation. The endotoxin content was required to be ≤0.1 EU / μg. Activity verification: This was verified using a Transwell chemotaxis assay. The specific steps were as follows: the tumor cells modified in step S1 were adjusted to a concentration of 1×10⁻⁶. 5Cells were seeded at a density of 1 / mL into the upper chamber of an 8μm pore size Transwell chamber, with 200μL of cell suspension added to each chamber. The lower chamber contained 600μL of culture medium with 50ng / mL SDF-1α. A control group was also included, with the lower chamber containing culture medium without SDF-1α. Cells were incubated at 37℃ and 5% CO2 for 5 hours. Unmigrated cells in the upper chamber were wiped off with a cotton swab, and the cells were stained with 0.1% crystal violet for 20 minutes, followed by rinsing three times with PBS. The number of migrating cells in five random fields was counted using ImageJ software under a 200x optical microscope. The cell viability retention rate was calculated based on the number of chemotactic cells in the control group: viability retention rate = (number of migrating cells in the experimental group / number of migrating cells in the control group) × 100%, with a requirement of ≥80%.

[0011] Preferably, if step S3 uses a sustained-release carrier implantation method to deliver SDF-1α, the sustained-release carrier is polylactic acid-glycolic acid copolymer microspheres, and the specific parameters and preparation method are as follows: PLGA parameters: The PLGA has a lactate-glycolic acid molar ratio of 50:50 and a molecular weight of 70,000-80,000. It was detected by gel permeation chromatography with tetrahydrofuran as the mobile phase and a flow rate of 1 mL / min. Microsphere parameters: Particle size 50-200 μm, measured by laser particle size analyzer; dispersion medium is deionized water; stirring speed 1500 rpm; drug loading 8-12%, measured by UV spectrophotometer at 280 nm wavelength; calculated according to SDF-1α standard curve, drug loading = (mass of SDF-1α in microspheres / total mass of microspheres) × 100%; release performance requires continuous release of SDF-1α for 10 days with daily release ≥ 8% of initial drug loading, activity retention ≥ 80%, and the activity of the released solution is detected by the Transwell chemotaxis experiment described in claim 5. The PLGA microspheres were prepared using an emulsion solvent evaporation method, and the specific steps included: Add dichloromethane at 15 times the mass of PLGA and stir magnetically at 300 rpm for 30 minutes until the PLGA is completely dissolved, forming an 8% (w / v) organic phase. SDF-1α was dissolved in sterile PBS at pH 7.2-7.4 as the aqueous phase. The mixture was prepared by mixing organic phase and aqueous phase at a volume ratio of 4:1 and placed in an ultrasonic disruptor for ultrasonic emulsification. The ultrasonic power was 250W and the ultrasonic time was 45 seconds. The ultrasonic mode was 3 seconds of operation followed by 5 seconds of interval to form the colostrum. The colostrum was slowly added to an aqueous solution containing 3% polyvinyl alcohol (polyvinyl alcohol with a molecular weight of 50,000). The volume ratio of colostrum to aqueous solution was 1:10. The solution was magnetically stirred at 600 rpm at 25°C for 4 hours to evaporate the solvent. Then, the microspheres were collected by centrifugation at 3500 rpm for 12 minutes and washed three times with deionized water, centrifuged for 5 minutes each time. The microspheres were placed in a freeze dryer and freeze-dried for 30 hours at a temperature of -55℃ and a vacuum degree of ≤10Pa to obtain dried microspheres. In vitro release experiment: 10 mg of microspheres were placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and 5 mL of sterile PBS was added to the dialysis bag. The dialysis bag was then placed in a centrifuge tube containing 50 mL of sterile PBS and shaken at 37 °C and 100 rpm. 5 mL samples were taken at 1, 2, 3, 7, and 10 days, and 5 mL of fresh PBS was added after each sample collection. The concentration of SDF-1α in the release solution was detected by an ELISA kit, and the cumulative release rate was calculated.

[0012] Preferably, the specific steps for in vivo imaging in step S4 are as follows: Animals were fasted for 12 hours before imaging, but were allowed free access to water during the fasting period; 15 mg / kg of D-fluorescein potassium salt was injected intraperitoneally, with an injection volume of 100 μL per animal. Imaging was performed 18 minutes after injection using a small animal fluorescence imaging system. The imaging parameters were set as follows: excitation wavelength 535nm, emission wavelength 580nm, exposure time 3 minutes, and field of view 10cm×10cm. Quantitative analysis was performed using LivingImage 4.7.3 software: The target metastatic organ (brain or liver) region was selected, and the fluorescence signal area and average optical density of the region were calculated. The relative volume of the metastatic lesion was calculated according to the formula: relative volume of metastatic lesion = (fluorescence signal area × average optical density) / 1000. Dividing by 1000 in the formula is a standardization process used to eliminate differences in instrument signals.

[0013] Preferably, the preparation and staining of histopathological sections in step S4 includes the following steps: S41. Tissue processing: After animal euthanasia, the target organ was harvested by cervical dislocation and fixed with 4% paraformaldehyde for 24 hours. Then, a gradient dehydration process was performed, using ethanol concentrations of 70%, 80%, 90%, 95%, and 100% successively, with each concentration treated for 1 hour, including two treatments with 100% ethanol. After dehydration, the tissue was cleared twice with xylene for 30 minutes each time. After clearing, the tissue was embedded in paraffin at 60°C for 2 hours (paraffin melting point is 56-58°C). 4μm thick sections were prepared using a microtome and mounted on poly-L-lysine-coated slides. S42. HE staining: Sections were baked in a 60℃ oven for 30 minutes, dewaxed twice with xylene for 10 minutes each time; then subjected to gradient rehydration with ethanol concentrations of 100%, 95%, 90%, 80%, and 70% successively, each concentration treated for 5 minutes, followed by washing with distilled water for 5 minutes; stained with hematoxylin for 5 minutes, followed by washing with tap water for 2 minutes; differentiated with 1% hydrochloric acid-ethanol (prepared by mixing 1 mL of hydrochloric acid and 99 mL of 70% ethanol) for 30 seconds, followed by washing with tap water for 5 minutes; stained with eosin for 2 minutes, followed by washing with tap water for 1 minute; dehydrated again with gradient dehydration with ethanol concentrations of 70%, 80%, 90%, 95%, and 100% successively, each concentration treated for 30 seconds, with 100% ethanol treated twice; cleared twice with xylene for 5 minutes each time; finally mounted with neutral resin. S43. Immunohistochemical staining: The dewaxing and rehydration steps are the same as in S42; incubate with 3% H2O2 at room temperature for 10 minutes, wash three times with distilled water for 5 minutes each time; perform antigen retrieval at pH 6.0 citrate buffer at 121℃ and 0.12 MPa for 2 minutes under high temperature and high pressure, cool naturally to room temperature, and wash three times with PBS for 5 minutes each time; block with 5% bovine serum albumin for 30 minutes, discard the blocking solution, and do not wash; add primary antibody for incubation, the anti-VEGF antibody is diluted 1:200, and the anti-MM antibody... The P-9 antibody was diluted 1:150 and incubated in a humidified chamber at 4°C for 16 hours. After incubation, the slides were washed three times with PBS for 5 minutes each time. Secondary antibody (HRP-labeled goat anti-rabbit IgG, diluted 1:5000) was added and incubated at room temperature for 30 minutes. After incubation, the slides were washed three times with PBS for 5 minutes each time. The reaction was terminated by developing the slides with DAB chromogenic solution for 4 minutes and washing with distilled water for 5 minutes. The slides were counterstained with hematoxylin for 2 minutes and washed with tap water for 5 minutes. Subsequent dehydration, clearing, and mounting procedures were the same as in S42. S44. Semi-quantitative scoring was performed using the H-score method, with five random fields of view observed under a 400x optical microscope. The H-score calculation formula is H-score = Σ positive cell percentage × staining intensity, where the positive cell percentage is divided into grade 0 (0%), grade 1 (1-25%), grade 2 (26-50%), grade 3 (51-75%), and grade 4 (76-100%), and the staining intensity is divided into grade 0 (none), grade 1 (weak), grade 2 (medium), and grade 3 (strong). An H-score ≥ 180 is considered high expression.

[0014] Compared with related technologies, the method for constructing a tumor animal model that simulates clinical metastasis characteristics provided by this invention has the following beneficial effects: 1. This invention provides a method for constructing a tumor animal model that simulates clinical metastasis characteristics. Through a dual mechanism of "precise modification of the CXCR4 gene + targeted regulation of the target organ microenvironment", it achieves highly efficient targeting of tumor metastasis: on the one hand, the key functional region of the CXCR4 gene is edited using CRISPR-Cas9 technology to steadily increase the receptor expression level by 2-5 times and enhance the chemotactic response ability of tumor cells; on the other hand, a specific chemotactic signal gradient is constructed for the target organ by local injection of recombinant human SDF-1α. The final model can achieve ≥5 metastatic lesions in the target organ / organ, and the metastasis rate in non-target organs is ≤10%. It strictly follows the clinical metastasis pattern and accurately replicates the clinical "primary lesion-target organ" metastasis path, completely improving the defects of traditional models such as random metastasis and poor target organ specificity.

[0015] 2. This invention provides a method for constructing a tumor animal model that simulates clinical metastatic characteristics. Cell pretreatment ensures "80-90% confluence after digestion and ≥90% viable cell ratio," while CRISPR-Cas9 operation specifies "sgRNA purity ≥98% and lentivirus titer ≥1×10⁻⁶." 8 The screening process uses a TU / mL and fluorescence intensity increase of 3-4 times. The detection process standardizes in vivo imaging, HE staining, and VEGF / MMP-9H-score semi-quantitative procedures. Multiple batch validations show a model success rate of ≥90%, with a coefficient of variation of ≤15% for the number of metastatic lesions within the same batch, significantly superior to traditional models. Simultaneously, a dual approach of "direct injection + PLGA microsphere sustained release" for SDF-1α is provided. The sustained-release microspheres can achieve continuous release for 10 days, reducing stress and operational errors in mice and further improving experimental stability.

[0016] 3. This invention provides a method for constructing a tumor animal model that simulates clinical metastatic characteristics. At the cellular level, it supports dual selection of "standardized cell lines + patient primary cells," taking into account both general research and personalized treatment needs. At the molecular level, it requires that the VEGF and MMP-9H-scores of metastatic lesions be increased by 1.5-3 times compared with the primary lesion, ensuring that the model matches the clinical tumor molecular mechanism. At the application level, it can cover metastasis mechanism research, anti-metastatic drug screening, and personalized treatment validation, providing a reliable animal model integrating "morphology-molecule-function" for clinical translational research, and solving the problem of the disconnect between traditional models and clinical pathological characteristics. Attached Figure Description

[0017] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0019] Example 1: Please see Figure 1 This invention provides a technical solution: a method for constructing a tumor animal model that simulates clinical metastasis characteristics, comprising the following steps: S1. Modification of CXCR4 chemokine receptor in tumor cells: Target tumor cells were modified using CRISPR-Cas9 technology. The editing target was the 120-150bp coding region of the NM_001308142.1 sequence of the CXCR4 gene. After modification, the expression level of CXCR4 increased by 2-5 times compared with that before modification. The results were detected by flow cytometry, with unmodified cells as the control. The specific operation of CRISPR-Cas9 technology in step S1 includes the following steps: sgRNA Design and Validation: An sgRNA targeting the CXCR4 gene was designed with a forward sequence of 5'-GGAATGGCGTGGATGAAGCG-3' and a reverse sequence of 5'-CGCTTCATCCACGCCATTCC-3'. The purity of the sgRNA was verified by 1% agarose gel electrophoresis, requiring a purity of ≥98%. Vector construction: The sgRNA and Cas9 protein-coding sequence were constructed into the lentiviral vector pLVX-EF1α-Puro. The GenBank accession number for the Cas9 protein-coding sequence is NM_001191186.1, and the Addgene number for the lentiviral vector pLVX-EF1α-Puro is 85132. During vector construction, the restriction endonuclease BsmBI was used for digestion at 37°C for 2 hours, followed by ligation with T4 DNA ligase at 22°C for 1 hour. The ligation product was transformed into DH5α competent cells, plated on LB agar plates containing ampicillin, and cultured at 37°C for 16 hours. Single clones were picked for sequencing verification. The primers used for sequencing were U6-F and U6-R. Lentiviral Packaging and Transfection: The sequencing-verified vector was transfected into 293T cells to package lentivirus. One day before transfection, 293T cells were seeded into 6-well plates at a seeding density of 2 × 10⁶ cells / well. 6Transfection was performed using vector and Lipofectamine 3000 1 μg: 2 μL at a ratio of 1 μg to 2 μL. The medium was incubated at 37°C for 6 hours, then replaced with fresh medium. Viral supernatant was collected 48 hours after transfection and filtered through a 0.45 μm filter. Viral titer was determined using the CCK-8 assay, requiring a titer ≥ 1 × 10⁻⁶. 8 TU / mL, titer calculation was based on the viral dilution at 50% viability of 293T cells; Cell selection: Lentiviral cells were added to the target tumor cell culture system at MOI=15, along with 8 μg / mL polybrene, and incubated at 37°C for 24 hours. After 48 hours of transfection, the medium was replaced with fresh medium. After 72 hours, the cells were selected with 2 μg / mL puromycin for 7 days, with the puromycin-containing medium being replaced daily during the selection period. Cell lines expressing high CXCR4 were selected by flow cytometry, with untransfected cells as a negative control. Cell lines with fluorescence intensity 3-4 times higher than the negative control were selected and cultured to the logarithmic growth phase for later use. In this implementation plan, sgRNA was designed targeting the 120-150bp coding region of the CXCR4 gene NM_001308142.1 sequence because this region is a key functional domain of the gene, and transcriptional regulatory sites can be precisely activated by CRISPR-Cas9 to ensure a stable 2-5 fold increase in CXCR4 expression after modification. The requirement of sgRNA purity ≥98% avoids cellular gene disorder caused by non-specific cleavage, while the viral titer ≥1×10⁻⁶ is also considered. 8 The settings of TU / mL and MOI=15 are based on preliminary experimental verification—these conditions enable lentiviral transfection efficiency to reach over 85%, while avoiding the inhibition of tumor cell activity by high-titer viruses; the criteria of 7 days of puromycin screening and a 3-4 fold increase in fluorescence intensity can accurately enrich cell lines that highly express CXCR4, laying the molecular basis for the subsequent directed migration of tumor cells to target organs.

[0020] S2. In situ seeding of modified tumor cells: The cells from step S1 were adjusted to a concentration using sterile PBS containing 0.2% trypan blue and pH 7.2-7.4, and then in situ seeded into the primary lesions of 6-8 week old NOD / SCID mice or BALB / c nude mice, specifically as follows: When the target organ for metastasis is the brain, and the primary lesion is the lung, intratracheal injection is used, with an injection depth of 2-3 mm and a cell seeding quantity of 1×10⁶ cells. 6 -5×10 6 Inoculation volume: 50-100 μL / animal; When the target metastatic organ is the liver and the primary lesion is the colon, submucosal injection into the intestinal wall is used, with an injection depth of 1-2 mm. The number and volume of cells inoculated are consistent with the brain metastasis model. The target tumor cells in step S2 are selected from any of the following: The non-small cell lung cancer cell line is A549, and its ATCC number is CCL-185. HCT116 colorectal cancer cells, ATCC designation CCL-247; The primary cells derived from the patient underwent the following processing: fresh lung cancer or colorectal cancer tissue with a volume ≥0.5 cm³ was obtained with the patient's informed consent; the tissue was washed three times with PBS containing penicillin (100 U / mL) and streptomycin (100 μg / mL) for 5 minutes each time; the tumor tissue was minced to less than 1 mm³, and 0.1% collagenase IV was added, followed by shaking digestion at 37°C and 120 rpm for 45 minutes; the cells were then filtered through a 70 μm cell sieve to obtain a single-cell suspension. The culture medium was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were then cultured in DMEM / F12 medium containing 15% fetal bovine serum and 2 ng / mLEGF at 37°C with 5% CO2. The medium was changed every 2-3 days during culture, and the number of passages was limited to no more than 5. The purity of the tumor marker CK18 was confirmed by flow cytometry, requiring a CK18-positive cell ratio ≥95%. Simultaneously, cell identity was verified by short tandem repeat (STR) sequence detection to ensure consistency between the STR typing and the original tumor tissue. In this implementation plan, the lungs and colon are selected as primary lesions for brain metastases and liver metastases, respectively, following the clinical patterns of tumor metastasis and making the model more closely resemble clinical pathological characteristics. Intratracheal injection depth is controlled at 2-3 mm, and submucosal injection depth in the intestinal wall is 1-2 mm to avoid damage to internal organs from excessive injection, while ensuring precise cell engraftment at the primary lesion. The seeded cell quantity is 1×10⁻⁶ cells / mL. 6 -5×10 6 The parameters of 50-100 μL / mouse were determined through multiple batches of preliminary experiments. This dosage can form a visible primary lesion within 2 weeks without causing premature death of mice due to excessive cell load. The selection of A549, HCT116 cell lines and patient primary cells can meet the research requirements of "standardized model" and "individualized model" respectively. Among them, the requirements of patient primary cells being passaged no more than 5 times and having a CK18 positive rate of ≥95% can preserve the genetic characteristics and metastatic potential of the original tumor to the greatest extent.

[0021] S3. Target organ microenvironment-directed regulation: Two days after inoculation, a specific ligand of CXCR4 was injected locally into the target transfer organ. The ligand was recombinant human SDF-1α, with a concentration of 50-80 ng / mL and a single injection volume of 30-40 μL / animal. The injection frequency was once every 2 days, for a total of 3 injections. The chemokine ligand SDF-1α in step S3 must meet the following validation criteria: Purity verification: SDS-PAGE electrophoresis was performed using a 12% separating gel at 100V for 90 minutes. After Coomassie Brilliant Blue staining, the proportion of the SDF-1α band gray value to the total band gray value was analyzed using ImageJ software. This proportion was required to be ≥95%. Endotoxin detection: The Limulus amebocyte lysate (LAL) reagent method was used for detection. The procedure was followed according to the kit instructions. The standard endotoxin concentration was used as a control for calculation. The endotoxin content was required to be ≤0.1 EU / μg. Activity verification: This was verified using a Transwell chemotaxis assay. The specific steps were as follows: the tumor cells modified in step S1 were adjusted to a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1 / mL in the upper chamber of an 8μm pore size Transwell chamber, with 200μL of cell suspension added to each chamber. The lower chamber contained 600μL of culture medium with 50ng / mL SDF-1α. A control group was also included, with the lower chamber containing culture medium without SDF-1α. Cells were incubated at 37℃ and 5% CO2 for 5 hours. Unmigrated cells in the upper chamber were wiped off with a cotton swab, and the cells were stained with 0.1% crystal violet for 20 minutes, followed by rinsing three times with PBS. The number of migrating cells in five random fields was counted using ImageJ software under a 200x optical microscope. The cell viability retention rate was calculated based on the number of chemotactic cells in the control group: viability retention rate = (number of migrating cells in the experimental group / number of migrating cells in the control group) × 100%, with a requirement of ≥80%. If step S3 uses a sustained-release carrier implantation method to deliver SDF-1α, and the sustained-release carrier is polylactic acid-glycolic acid copolymer microspheres, the specific parameters and preparation method are as follows: PLGA parameters: The PLGA has a lactate-glycolic acid molar ratio of 50:50 and a molecular weight of 70,000-80,000. It was detected by gel permeation chromatography with tetrahydrofuran as the mobile phase and a flow rate of 1 mL / min. Microsphere parameters: Particle size 50-200 μm, measured by laser particle size analyzer; dispersion medium is deionized water; stirring speed 1500 rpm; drug loading 8-12%, measured by UV spectrophotometer at 280 nm wavelength; calculated according to SDF-1α standard curve, drug loading = (mass of SDF-1α in microspheres / total mass of microspheres) × 100%; release performance requires continuous release of SDF-1α for 10 days with daily release ≥ 8% of initial drug loading, activity retention ≥ 80%, and the activity of the released solution is detected by the Transwell chemotaxis experiment of claim 5. The PLGA microspheres were prepared using an emulsion solvent evaporation method, and the specific steps included: Add dichloromethane at 15 times the mass of PLGA and stir magnetically at 300 rpm for 30 minutes until the PLGA is completely dissolved, forming an 8% (w / v) organic phase. SDF-1α was dissolved in sterile PBS at pH 7.2-7.4 as the aqueous phase. The mixture was prepared by mixing organic phase and aqueous phase at a volume ratio of 4:1 and placed in an ultrasonic disruptor for ultrasonic emulsification. The ultrasonic power was 250W and the ultrasonic time was 45 seconds. The ultrasonic mode was 3 seconds of operation followed by 5 seconds of interval to form the colostrum. The colostrum was slowly added to an aqueous solution containing 3% polyvinyl alcohol (polyvinyl alcohol with a molecular weight of 50,000). The volume ratio of colostrum to aqueous solution was 1:10. The solution was magnetically stirred at 600 rpm at 25°C for 4 hours to evaporate the solvent. Then, the microspheres were collected by centrifugation at 3500 rpm for 12 minutes and washed three times with deionized water, centrifuged for 5 minutes each time. The microspheres were placed in a freeze dryer and freeze-dried for 30 hours at a temperature of -55℃ and a vacuum degree of ≤10Pa to obtain dried microspheres. In vitro release assay: 10 mg of microspheres were placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and 5 mL of sterile PBS was added to the dialysis bag. The dialysis bag was then placed in a centrifuge tube containing 50 mL of sterile PBS and the mixture was shaken at 37 °C and 100 rpm. 5 mL samples were taken at 1, 2, 3, 7, and 10 days, and 5 mL of fresh PBS was added after each sample collection. The concentration of SDF-1α in the release solution was detected using an ELISA kit, and the cumulative release rate was calculated. In this implementation plan, recombinant human SDF-1α was chosen as the CXCR4-specific ligand because the two have a high affinity for each other, which can efficiently activate the chemotactic signaling pathway in tumor cells. The SDF-1α concentration of 50-80 ng / mL and the single injection volume of 30-40 μL per animal are based on the concentration calculation of the local microenvironment of the target organ—this dose can maintain the local ligand concentration of the target organ at 10-15 ng / mL and avoid metastasis to non-target organs caused by systemic spread. The frequency of injection once every 2 days for a total of 3 times can cover the period from tumor cell shedding from the primary lesion to migration to other sites. The critical window period of the target organ further enhances the targeted transfer effect. If PLGA microspheres are used for delivery, the choice of a lactate-glycolic acid molar ratio of 50:50 is because the degradation cycle of PLGA in vivo is about 10 days, which can match the continuous release requirements of SDF-1α. The particle size of 50-200μm can be retained around the target organ through local implantation, avoiding the loss of microspheres with body fluid circulation. The standard of drug loading of 8-12% and daily release of ≥8% can ensure the stability of local ligand concentration in the target organ within 10 days, and the activity retention rate of ≥80% ensures that the ligand always has chemotactic activity.

[0022] S4. Model Cultivation and Validation: Animals treated in step S3 were cultured for 3 weeks in an SPF-grade environment with a temperature of 22-25℃, humidity of 40-60%, and a 12-hour light-dark cycle. Animals were weighed weekly during the cultivation period. Fluorescence signals in the target metastatic organs were detected using in vivo imaging, and the number of metastatic lesions was counted using HE staining. Immunohistochemistry was used to detect the expression levels of VEGF and MMP-9 in the metastatic lesions. A model was considered a qualified model if the following conditions were met: The number of metastatic lesions in the target organ is ≥5 per organ, and the metastasis rate in non-target organs is ≤10%; The H-score values ​​of VEGF and MMP-9 in metastatic lesions were 1.5-3 times higher than those in primary lesions. The H-score was quantitatively calculated using ImageJ software. The staining intensity was divided into three grades: weak, medium, and strong according to the OD value. The proportion of positive cells was graded according to the counting results. The specific steps for live imaging in step S4 are as follows: Animals were fasted for 12 hours before imaging, but were allowed free access to water during the fasting period; 15 mg / kg of D-fluorescein potassium salt was injected intraperitoneally, with an injection volume of 100 μL per animal. Imaging was performed 18 minutes after injection using a small animal fluorescence imaging system. The imaging parameters were set as follows: excitation wavelength 535nm, emission wavelength 580nm, exposure time 3 minutes, and field of view 10cm×10cm. Quantitative analysis was performed using LivingImage 4.7.3 software: The target metastatic organ region was selected, and the fluorescence signal area and average optical density of the region were calculated. The relative volume of the metastatic lesion was calculated according to the formula: (fluorescence signal area × average optical density) / 1000. Dividing by 1000 in the formula is a standardization process to eliminate differences in instrument signals. Step S4, the preparation and staining of histopathological sections, includes the following steps: S41. Tissue processing: After animal euthanasia, the target organ was harvested by cervical dislocation and fixed with 4% paraformaldehyde for 24 hours. Then, a gradient dehydration process was performed, using ethanol concentrations of 70%, 80%, 90%, 95%, and 100% successively, with each concentration treated for 1 hour, including two treatments with 100% ethanol. After dehydration, the tissue was cleared twice with xylene for 30 minutes each time. After clearing, the tissue was embedded in paraffin at 60°C for 2 hours (paraffin melting point is 56-58°C). 4μm thick sections were prepared using a microtome and mounted on poly-L-lysine-coated slides. S42. HE staining: Sections were baked in a 60℃ oven for 30 minutes, dewaxed twice with xylene for 10 minutes each time; then subjected to gradient rehydration with ethanol concentrations of 100%, 95%, 90%, 80%, and 70% successively, each concentration treated for 5 minutes, followed by washing with distilled water for 5 minutes; stained with hematoxylin for 5 minutes, followed by washing with tap water for 2 minutes; differentiated with 1% hydrochloric acid-ethanol (prepared by mixing 1 mL of hydrochloric acid and 99 mL of 70% ethanol) for 30 seconds, followed by washing with tap water for 5 minutes; stained with eosin for 2 minutes, followed by washing with tap water for 1 minute; dehydrated again with gradient dehydration with ethanol concentrations of 70%, 80%, 90%, 95%, and 100% successively, each concentration treated for 30 seconds, with 100% ethanol treated twice; cleared twice with xylene for 5 minutes each time; finally mounted with neutral resin. S43. Immunohistochemical staining: The dewaxing and rehydration steps are the same as in S42; incubate with 3% H2O2 at room temperature for 10 minutes, wash three times with distilled water for 5 minutes each time; perform antigen retrieval at pH 6.0 citrate buffer at 121℃ and 0.12 MPa for 2 minutes under high temperature and high pressure, cool naturally to room temperature, and wash three times with PBS for 5 minutes each time; block with 5% bovine serum albumin for 30 minutes, discard the blocking solution, and do not wash; add primary antibody for incubation, the anti-VEGF antibody is diluted 1:200, and the anti-MM antibody... The P-9 antibody was diluted 1:150 and incubated in a humidified chamber at 4°C for 16 hours. After incubation, the slides were washed three times with PBS for 5 minutes each time. Secondary antibody (HRP-labeled goat anti-rabbit IgG, diluted 1:5000) was added and incubated at room temperature for 30 minutes. After incubation, the slides were washed three times with PBS for 5 minutes each time. The reaction was terminated by developing the slides with DAB chromogenic solution for 4 minutes and washing with distilled water for 5 minutes. The slides were counterstained with hematoxylin for 2 minutes and washed with tap water for 5 minutes. Subsequent dehydration, clearing, and mounting procedures were the same as in S42. S44. Semi-quantitative scoring was performed using the H-score method, with five random fields of view observed under a 400x optical microscope. The H-score calculation formula is H-score = Σ positive cell percentage × staining intensity, where the positive cell percentage is divided into grade 0 (0%), grade 1 (1-25%), grade 2 (26-50%), grade 3 (51-75%), and grade 4 (76-100%), and the staining intensity is divided into grade 0 (none), grade 1 (weak), grade 2 (medium), and grade 3 (strong). An H-score ≥ 180 was considered high expression. In this implementation plan, the SPF-grade ambient temperature of 22-25℃, humidity of 40-60%, and 12-hour light-dark cycle settings follow experimental animal husbandry standards, avoiding the impact of external microbial infections and environmental stress on the physiological state of mice and ensuring model stability. Weighing mice once a week allows for assessment of their health status through weight changes, eliminating experimental errors caused by non-tumor factors. Fasting for 12 hours before in vivo imaging reduces fluorescence interference from intestinal contents. Intraperitoneal injection of 15 mg / kg D-fluorescein potassium followed by imaging 18 minutes later is due to the dosage and timing... The application of fluorescein allows for a full reaction between fluorescein and luciferase within tumor cells, resulting in a peak fluorescence signal. The 30-second differentiation step with 1% hydrochloric acid ethanol in HE staining precisely controls the hematoxylin staining depth, preventing excessive nuclear staining from obscuring details of metastatic lesions. High-temperature, high-pressure antigen retrieval using pH 6.0 citrate buffer in immunohistochemistry effectively exposes VEGF and MMP-9 epitopes, increasing antibody binding efficiency by over 40%. An H-score ≥ 180 is used to determine high expression, referencing clinical tumor metastasis pathology data to ensure consistency between the model and clinical metastasis characteristics.

[0023] The process also includes tumor cell pretreatment, specifically: target tumor cells are seeded in high-glucose DMEM medium containing 10% mycoplasma-negative fetal bovine serum and 1% penicillin-streptomycin, with penicillin at a concentration of 100 U / mL and streptomycin at a concentration of 100 μg / mL; the culture environment is a constant temperature incubator at 37℃ and 5% CO2; when the cell confluence reaches 80-90%, the cells are digested with 0.25% trypsin-EDTA digestion solution at 37℃ for 2.5 minutes; then centrifuged at 1100 rpm for 6 minutes, and the cells are collected and resuspended in sterile PBS buffer at pH 7.2-7.4; the cell concentration is adjusted to 5 × 10⁶ cells / mL. 7 -8×10 7 Cell viability was measured by trypan blue staining, with a live cell ratio of ≥90%; cells were then used after meeting the standard. In this implementation plan, a high-glucose DMEM medium containing 10% mycoplasma-negative fetal bovine serum is used because mycoplasma contamination can lead to disordered gene expression in tumor cells, while a high-glucose environment can meet the high metabolic needs of tumor cells and maintain their proliferative activity. The penicillin-streptomycin dual antibiotic combination can effectively inhibit contamination by Gram-positive and Gram-negative bacteria and has extremely low toxicity to tumor cells. Digestion is performed when the cell confluence reaches 80-90% because the cells are in the logarithmic growth phase at this stage, with strong proliferative capacity and stable metastatic potential, avoiding the impact of too low or too high confluence on subsequent experiments. Treatment with 0.25% trypsin-EDTA digestion solution at 37°C for 2.5 minutes can avoid cell membrane damage caused by excessive enzymatic digestion while digesting cells. Centrifugation at 1100 rpm for 6 minutes can fully precipitate cells without destroying cell integrity. The cell concentration is adjusted to 5 × 10⁶ cells / year.7 -8×10 7 The requirement of cells / mL and a viable cell ratio of ≥90% ensures uniform cell density during subsequent inoculation and reduces the failure of primary lesion formation due to insufficient cell viability.

[0024] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, the phrase "comprising an element defined as..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0025] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a tumor animal model that simulates clinical metastasis characteristics, characterized in that: Includes the following steps: S1. Modification of CXCR4 chemokine receptor in tumor cells: Target tumor cells were modified using CRISPR-Cas9 technology. The editing target was the 120-150bp coding region of the NM_001308142.1 sequence of the CXCR4 gene. After modification, the expression level of CXCR4 increased by 2-5 times compared with that before modification. The results were detected by flow cytometry, with unmodified cells as the control. S2. In situ seeding of modified tumor cells: The cells from step S1 were adjusted to a concentration using sterile PBS containing 0.2% trypan blue and pH 7.2-7.4, and then in situ seeded into the primary lesions of 6-8 week old NOD / SCID mice or BALB / c nude mice, specifically as follows: When the target organ for metastasis is the brain, and the primary lesion is the lung, intratracheal injection is used, with an injection depth of 2-3 mm and a cell seeding quantity of 1×10⁶ cells. 6 -5×10 6 Inoculation volume: 50-100 μL / animal; When the target metastatic organ is the liver and the primary lesion is the colon, submucosal injection into the intestinal wall is used, with an injection depth of 1-2 mm. The number and volume of cells inoculated are consistent with the brain metastasis model. S3. Target organ microenvironment-directed regulation: Two days after inoculation, a specific ligand of CXCR4 was injected locally into the target transfer organ. The ligand was recombinant human SDF-1α, with a concentration of 50-80 ng / mL and a single injection volume of 30-40 μL / animal. The injection frequency was once every 2 days, for a total of 3 injections. S4. Model cultivation and verification: Animals treated in step S3 were placed in an SPF-grade environment with a temperature of 22-25℃, humidity of 40-60%, and 12h light-dark cycle for 3 weeks. During the cultivation period, the animals were weighed once a week. Fluorescence signals in target metastatic organs were detected by in vivo imaging, and the number of metastatic lesions was counted by HE staining. The expression levels of VEGF and MMP-9 in metastatic lesions were detected by immunohistochemistry. A model was deemed qualified if the following conditions were met: The number of metastatic lesions in the target organ is ≥5 per organ, and the metastasis rate in non-target organs is ≤10%; The H-score values ​​of VEGF and MMP-9 in metastatic lesions were 1.5-3 times higher than those in primary lesions. The H-score was quantitatively calculated using ImageJ software. The staining intensity was divided into three grades: weak, medium, and strong according to the OD value. The proportion of positive cells was graded according to the counting results.

2. The method for constructing a tumor animal model simulating clinical metastasis characteristics according to claim 1, characterized in that: The process also includes tumor cell pretreatment, specifically: target tumor cells are seeded in high-glucose DMEM medium containing 10% mycoplasma-negative fetal bovine serum and 1% penicillin-streptomycin, with penicillin at a concentration of 100 U / mL and streptomycin at a concentration of 100 μg / mL; the culture environment is a constant temperature incubator at 37℃ and 5% CO2; when the cell confluence reaches 80-90%, the cells are digested with 0.25% trypsin-EDTA digestion solution at 37℃ for 2.5 minutes; then centrifuged at 1100 rpm for 6 minutes, and the cells are collected and resuspended in sterile PBS buffer at pH 7.2-7.4; the cell concentration is adjusted to 5 × 10⁶ cells / mL. 7 -8×10 7 Cell viability was measured by trypan blue staining, with a requirement of ≥90% live cells. Cells were then used for future reference.

3. The method for constructing a tumor animal model simulating clinical metastasis characteristics according to claim 1, characterized in that: The specific operation of CRISPR-Cas9 technology in step S1 includes the following steps: sgRNA Design and Validation: An sgRNA targeting the CXCR4 gene was designed with a forward sequence of 5'-GGAATGGCGTGGATGAAGCG-3' and a reverse sequence of 5'-CGCTTCATCCACGCCATTCC-3'. The purity of the sgRNA was verified by 1% agarose gel electrophoresis, requiring a purity of ≥98%. Vector construction: The sgRNA and Cas9 protein-coding sequence were constructed into the lentiviral vector pLVX-EF1α-Puro. The GenBank accession number for the Cas9 protein-coding sequence is NM_001191186.1, and the Addgene number for the lentiviral vector pLVX-EF1α-Puro is 85132. During vector construction, the restriction endonuclease BsmBI was used for digestion at 37°C for 2 hours, followed by ligation with T4 DNA ligase at 22°C for 1 hour. The ligation product was transformed into DH5α competent cells, plated on LB agar plates containing ampicillin, and cultured at 37°C for 16 hours. Single clones were picked for sequencing verification. The primers used for sequencing were U6-F and U6-R. Lentiviral Packaging and Transfection: The sequencing-verified vector was transfected into 293T cells to package lentivirus. One day before transfection, 293T cells were seeded into 6-well plates at a seeding density of 2 × 10⁶ cells / well. 6 Transfection was performed using vector and Lipofectamine 3000 1 μg: 2 μL at a ratio of 1 μg to 2 μL. The medium was incubated at 37°C for 6 hours, then replaced with fresh medium. Viral supernatant was collected 48 hours after transfection and filtered through a 0.45 μm filter. Viral titer was determined using the CCK-8 assay, requiring a titer ≥ 1 × 10⁻⁶. 8 TU / mL, titer calculation was based on the viral dilution at 50% viability of 293T cells; Cell selection: Lentiviral cells were added to the target tumor cell culture system at MOI=15, along with 8 μg / mL polybrene, and incubated at 37°C for 24 hours. After 48 hours of transfection, the medium was replaced with fresh medium. After 72 hours, the cells were selected with 2 μg / mL puromycin for 7 days, with the puromycin-containing medium being replaced daily during the selection period. Cell lines expressing high CXCR4 were selected by flow cytometry, with untransfected cells as a negative control. Cell lines with fluorescence intensity 3-4 times higher than the negative control were selected and cultured to the logarithmic growth phase for later use.

4. The method for constructing a tumor animal model simulating clinical metastasis characteristics according to claim 1, characterized in that: The target tumor cells in step S2 are selected from any of the following: The non-small cell lung cancer cell line is A549, and its ATCC number is CCL-185. HCT116 colorectal cancer cells, ATCC designation CCL-247; The patient-derived primary cells were processed by taking fresh lung cancer or colorectal cancer tissue with a volume ≥0.5 cm³, with the patient's informed consent obtained from the tissue. Wash the tumor tissue three times with PBS containing penicillin (100 U / mL) and streptomycin (100 μg / mL) for 5 minutes each time. Mince the tumor tissue to less than 1 mm³, add 0.1% collagenase IV, and digest with shaking at 37°C and 120 rpm for 45 minutes. Filter the cell suspension through a 70 μm cell sieve to obtain a single-cell suspension, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Culture the cells in DMEM / F12 medium containing 15% fetal bovine serum and 2 ng / mLEGF at 37°C and 5% CO2. Change the medium every 2-3 days during culture, and passage no more than 5 times. Confirm the purity of the tumor marker CK18 by flow cytometry, requiring a CK18 positive cell ratio ≥95%. Simultaneously, verify cell identity by short tandem repeat (STR) sequence detection to ensure that the STR typing is consistent with the original tumor tissue.

5. The method for constructing a tumor animal model simulating clinical metastasis characteristics according to claim 1, characterized in that: The chemokine ligand SDF-1α in step S3 must meet the following validation criteria: Purity verification: SDS-PAGE electrophoresis was performed using a 12% separating gel at 100V for 90 minutes. After Coomassie Brilliant Blue staining, the proportion of the SDF-1α band gray value to the total band gray value was analyzed using ImageJ software. This proportion was required to be ≥95%. Endotoxin detection: The Limulus amebocyte lysate (LAL) reagent method was used for detection. The procedure was followed according to the kit instructions. The standard endotoxin concentration was used as a control for calculation. The endotoxin content was required to be ≤0.1 EU / μg. Activity verification: This was verified using a Transwell chemotaxis assay. The specific steps were as follows: the tumor cells modified in step S1 were adjusted to a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 100 cells / mL into the upper chamber of an 8μm pore size Transwell chamber, with 200μL of cell suspension added to each chamber. 600μL of culture medium containing 50ng / mL SDF-1α was added to the lower chamber. A control group was also included, with culture medium without SDF-1α added to the lower chamber. Cells were incubated at 37℃ and 5% CO2 for 5 hours. Unmigrated cells from the upper chamber were wiped off with a cotton swab, and the cells were stained with 0.1% crystal violet for 20 minutes, followed by rinsing three times with PBS. The number of migrating cells in five random fields of view was counted using ImageJ software under an optical microscope with a 200x magnification. Using the number of chemotactic cells in the control group as a baseline, the viability retention rate was calculated as follows: viability retention rate = (number of migrating cells in the experimental group / number of migrating cells in the control group) × 100%, with a viability retention rate required to be ≥ 80%.

6. The method for constructing a tumor animal model simulating clinical metastasis characteristics according to claim 1, characterized in that: If step S3 uses a sustained-release carrier implantation method to deliver SDF-1α, the sustained-release carrier is polylactic acid-glycolic acid copolymer microspheres, and the specific parameters and preparation method are as follows: PLGA parameters: The PLGA has a lactate-glycolic acid molar ratio of 50:50 and a molecular weight of 70,000-80,000. It was detected by gel permeation chromatography with tetrahydrofuran as the mobile phase and a flow rate of 1 mL / min. Microsphere parameters: Particle size 50-200 μm, measured by laser particle size analyzer; dispersion medium is deionized water; stirring speed 1500 rpm; drug loading 8-12%, measured by UV spectrophotometer at 280 nm wavelength; calculated according to SDF-1α standard curve, drug loading = (mass of SDF-1α in microspheres / total mass of microspheres) × 100%; release performance requires continuous release of SDF-1α for 10 days with daily release ≥ 8% of initial drug loading, activity retention ≥ 80%, and the activity of the released solution is detected by the Transwell chemotaxis experiment described in claim 5. The PLGA microspheres were prepared using an emulsion solvent evaporation method, and the specific steps included: Add dichloromethane at 15 times the mass of PLGA and stir magnetically at 300 rpm for 30 minutes until the PLGA is completely dissolved, forming an 8% (w / v) organic phase. SDF-1α was dissolved in sterile PBS at pH 7.2-7.4 as the aqueous phase. The mixture was prepared by mixing organic phase and aqueous phase at a volume ratio of 4:1 and placed in an ultrasonic disruptor for ultrasonic emulsification. The ultrasonic power was 250W and the ultrasonic time was 45 seconds. The ultrasonic mode was 3 seconds of operation followed by 5 seconds of interval to form the colostrum. The colostrum was slowly added to an aqueous solution containing 3% polyvinyl alcohol (polyvinyl alcohol with a molecular weight of 50,000). The volume ratio of colostrum to aqueous solution was 1:

10. The solution was magnetically stirred at 600 rpm at 25°C for 4 hours to evaporate the solvent. Then, the microspheres were collected by centrifugation at 3500 rpm for 12 minutes and washed three times with deionized water, centrifuged for 5 minutes each time. The microspheres were placed in a freeze dryer and freeze-dried for 30 hours at a temperature of -55℃ and a vacuum degree of ≤10Pa to obtain dried microspheres. In vitro release experiment: 10 mg of microspheres were placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and 5 mL of sterile PBS was added to the dialysis bag. The dialysis bag was then placed in a centrifuge tube containing 50 mL of sterile PBS and shaken at 37 °C and 100 rpm. 5 mL samples were taken at 1, 2, 3, 7, and 10 days, and 5 mL of fresh PBS was added after each sample collection. The concentration of SDF-1α in the release solution was detected by an ELISA kit, and the cumulative release rate was calculated.

7. The method for constructing a tumor animal model simulating clinical metastasis characteristics according to claim 1, characterized in that: The specific steps for live imaging in step S4 are as follows: Animals were fasted for 12 hours before imaging, but were allowed free access to water during the fasting period; 15 mg / kg of D-fluorescein potassium salt was injected intraperitoneally, with an injection volume of 100 μL per animal. Imaging was performed 18 minutes after injection using a small animal fluorescence imaging system. The imaging parameters were set as follows: excitation wavelength 535nm, emission wavelength 580nm, exposure time 3 minutes, and field of view 10cm×10cm. Quantitative analysis was performed using LivingImage 4.7.3 software: The target metastatic organ region was selected, and the fluorescence signal area and average optical density of the region were calculated. The relative volume of the metastatic lesion was calculated according to the formula: relative volume of metastatic lesion = (fluorescence signal area × average optical density) / 1000. Dividing by 1000 in the formula is a standardization process used to eliminate differences in instrument signals.

8. The method for constructing a tumor animal model simulating clinical metastasis characteristics according to claim 1, characterized in that: Step S4, the preparation and staining of histopathological sections, includes the following steps: S41. Tissue processing: After animal euthanasia, the target organ was harvested by cervical dislocation and fixed with 4% paraformaldehyde for 24 hours. Then, a gradient dehydration process was performed, using ethanol concentrations of 70%, 80%, 90%, 95%, and 100% successively, with each concentration treated for 1 hour, including two treatments with 100% ethanol. After dehydration, the tissue was cleared twice with xylene for 30 minutes each time. After clearing, the tissue was embedded in paraffin at 60°C for 2 hours (paraffin melting point is 56-58°C). 4μm thick sections were prepared using a microtome and mounted on poly-L-lysine-coated slides. S42. HE staining: Sections were baked in a 60℃ oven for 30 minutes, dewaxed twice with xylene for 10 minutes each time; then subjected to gradient rehydration with ethanol concentrations of 100%, 95%, 90%, 80%, and 70% successively, each concentration treated for 5 minutes, followed by washing with distilled water for 5 minutes; stained with hematoxylin for 5 minutes, followed by washing with tap water for 2 minutes; differentiated with 1% hydrochloric acid-ethanol (prepared by mixing 1 mL of hydrochloric acid and 99 mL of 70% ethanol) for 30 seconds, followed by washing with tap water for 5 minutes; stained with eosin for 2 minutes, followed by washing with tap water for 1 minute; dehydrated again with gradient dehydration with ethanol concentrations of 70%, 80%, 90%, 95%, and 100% successively, each concentration treated for 30 seconds, with 100% ethanol treated twice; cleared twice with xylene for 5 minutes each time; finally mounted with neutral resin. S43. Immunohistochemical staining: The dewaxing and rehydration steps are the same as in S42; incubate with 3% H2O2 at room temperature for 10 minutes, wash three times with distilled water for 5 minutes each time; perform antigen retrieval at pH 6.0 citrate buffer at 121℃ and 0.12 MPa for 2 minutes under high temperature and high pressure, cool naturally to room temperature, and wash three times with PBS for 5 minutes each time; block with 5% bovine serum albumin for 30 minutes, discard the blocking solution, and do not wash; add primary antibody for incubation, the anti-VEGF antibody is diluted 1:200, and the anti-MM antibody... The P-9 antibody was diluted 1:150 and incubated in a humidified chamber at 4°C for 16 hours. After incubation, the slides were washed three times with PBS for 5 minutes each time. Secondary antibody (HRP-labeled goat anti-rabbit IgG, diluted 1:5000) was added and incubated at room temperature for 30 minutes. After incubation, the slides were washed three times with PBS for 5 minutes each time. The reaction was terminated by developing the slides with DAB chromogenic solution for 4 minutes and washing with distilled water for 5 minutes. The slides were counterstained with hematoxylin for 2 minutes and washed with tap water for 5 minutes. Subsequent dehydration, clearing, and mounting procedures were the same as in S42. S44. Semi-quantitative scoring was performed using the H-score method, with five random fields of view observed under a 400x optical microscope. The H-score calculation formula is H-score = Σ positive cell percentage × staining intensity, where the positive cell percentage is divided into grade 0 (0%), grade 1 (1-25%), grade 2 (26-50%), grade 3 (51-75%), and grade 4 (76-100%), and the staining intensity is divided into grade 0 (none), grade 1 (weak), grade 2 (medium), and grade 3 (strong). An H-score ≥ 180 is considered high expression.

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